Biotin aliphatic amides and method for their preparation



' paring the same.

Patented Aug. 22, 1950 BIOTIN A LIPI IATI-C AMIDES AND METHOD FOR THEIRPREPARATION Donald E. Wolf, Franklin Township, Somerset County," andKarl Folkers, Plainfield, N. J assignors to Merck & 00., Inc., Rahway, acorporation of New Jersey :No Drawing. Application June at, 1947, SerialNo.756,1-02 p a 18 Claims.

This invention is concerned generally with novel chemical compounds andprocesses ofpre- More particularly, it relates to (-01. zoo-s09) reactedwith an aliphatic amine in the presence novel biotin derivatives whichpossess physiological activity as growth promoting factors formicroorgamsms.

The novel chemical compounds forming the subject matter of the presentinvention are the biotin aliphatic amines. ,It has been established withreasonable certainty that these novel compounds have the followingstructuralformula:

I HN--NH Ht- EH H2(E-S JH(QH2)4 COR wherein R represents aliphatic aminoacids and aliphatic amino acid esters.

It has now been discovered inaccordance with the present invention thatthese new compounds may be prepared by reacting aliphatic amines withcompounds represented by the formula:

wherein R represents halogen and alkoxy substituents.

Starting materials wherein R in the above formula is a halogensubstituent are fully defined by the name biotin acid halides and d1-biotin acid halides wherein the carboxyl groups are converted to acidhalide groups. These biotin acid halide compounds can beprepar'ed byreacting biotin with phosphorus halides, sulfuryl halides and thionylhalides. This process and the products thereby obtained are fullydisclosed and claimed in a copending joint application by the presentinventors, Folkers, Mozingo and Wolf, Serial No. 554,4;60, filedSeptember 16, 1944, now Patent No. 2,442,681.

Compounds of the above formula wherein R represents alkoxy substituents'are fully defined by the name biotin alkyl esters and dl-biotin alkylesters. i

The aliphatic amines, which maybe employed as reactants in the presentinvention, include aliphatic amino acids and aliphatic amino acidesters. Compounds such as" glycine, and esters of alanine, leucine,asparticand'glutamic acids have been found'to .be satisfactory.

.In carrying out the process of the present invention abiotinjacidhalide or a biotin ester is of acondensing agent such aspyridine or sodium hydroxide. The reaction is preferably conducted atroom temperature, for a time sufiicient to effect ,condensation. Whensodium hydroxide is employed as the condensing agent, the solution canbe acidified with an inorganic acid, such as hydrochloric acid,whereupon the substituted biotin amide crystallizes from solution.Condensing agents such as pyridine are removed by evaporasubstitutedbiotin amide and impurities. .The

biotin. amide can be extracted with chloroform,

tion. The residue thus obtained is a mixture of the extract dried andthe chloroform removed-by oil. mixture diluted with water, whitecrystals of substituted biotin amide separate from solution.

Thecondensation of biotin and aliphatic amino ,,,acid esters results inthe formation of the biotinyl aliphatic amino acid ester. These esterscan be converted to biotinyl aliphatic: aminoacids by Of 2135-2145 C.

hydrolyzing the ester group. This is--accomevaporation leaving thebiotin amide as a clear Whenthe oil is dissolved in alcohol and theplished by heating the biotinyl aliphatic 'amine v ester with sodiumhydroxideat C., cooling the solution and adding an acid to precipitatethe biotinyl aliphatic amino acid.

The following examples illustrate a method of carrying out the presentinvention but it isto be 1 understood that these examples are givenbywayof illustration and not of limitation.

Example 1 To 24 mg. of biotin was added about '1 drops of l thionylchloride. The biotin dissolved and biotin -acid chloride crystallizedfrom solution. The eX--'- cess thionyl chloride was evaporated in vacuo.To thebiotin acid chloride, thusobtained, was 1 added a solution of 76mg. of glycine in 1 ml. of

sodium hydroxide (N) at 0". C. it After' thirty minutes the solution was-acidified,with 3 N hydro-- chloric acid and biotinyl glycineprecipitated as a white solid. The crystals, which were removed andrecrystallized from water, had

a meltingfpoint Anal. Calcd. for C12H19N304SZ c, 47.82; H, 6.35.

Found: C, 47.95; H, 6.90.

Example 2 mg. of biotin was converted to the To Q .5 ml. of fl-alanineethyl ester. The mixturewas allowed to stand at room temperature foreighteen f ;hours.. The v reaction product was dissolved.

3 about 15 cc. of .5 N hydrochloric acid and the solution extractedcontinuously with chloroform for two hours. The chloroform wasevaporated leaving biotinyl-c-alanine ethyl ester as a white solidmelting at 3132-135 Anal. Calcd. for C15H25N3O4S: "C, 52.47; H, 7.34; N,12.24. Found: C, 52.40; H, 7.29; N, 12.81.

To about 100 mg. of biotinyl-c-alanine ethyl ester was added ml. of 0.5N sodium hydroxide. The mixture was heated at 60 C. for twenty minutes.The solution was cooled and acidified with hydrochloric acid. Biotinylfl alanine separated as a white fiocculent precipitate and was collectedand washed on a filter. The product had a melting point of 229-232 C.Biotinyl-B- alanine was dried at 140 C. for analysis. 7

Anal. Calcd. for C13H21N3O4S2 C, 49.51; H, 6.71 N, 13.32. Found: C,49.26; H, 6.43; N, 13.01.

Example 3 About 100 mg. of biotin was converted to the acid chloride bytreatment with thionyl chloride. To the biotin acid chloride thusobtained was added 'a solution of 126 mg. of l(-)-leucine ethyl esterhydrochloride in about '2 ml. of pyridine. The reaction inixtii're wasallowed to stand at room temperature overnight and then concentrated invacuo. The residue was dissolved in chloroform and the chloroform washedwith dilute acid, bicarbonate, and water. The chlorofo'ri'n solution wasdried over magnesium sulfate and concentrated leavingbiotinyl-l()leu'cine ethyl ester as a clear oil. When dissolved in ethylalcohol, and diluted with water White crystals of biot'ihyl lbl'leiicine ethyl ester formed. These crystals which were recovered anddried, had a melting point of 116 0.

Anal. baled. for omihimois: c, 56.08; 8.11; N, "1on0. Found: 'c, 56.09;H, 8.16; 10.74.

Example 4 100 mg. or biotin was converted to biotin acid chloride bytreating it with -thionyl chloride. T the biotin acid chloride thusobtained was added a solution lea mg. or 'dl aspartic dieth'yl ester rin 1ml. of dry pyridine. The mixtu e was agitated Until the biotin acidchloride had dissolved. The 36111171611 'QHO'Wd to Stand at i'OOIl'ltemer-ature for 1% hours and was then evaporated at reduced pressureresulting in an oil containing ethyl biotinyl dl aspartate. The oil wasdissolved in chloroform and the chloroform solution Washed with diluteacid, bicarbonate, and Water. The chloroform solution of ethylbiotin'yl-dl-asartate was dried over magnesium sulfate and thechloroform evaporated. The residue was dissolved in ethanol and dilutedwith water whereupon ethyl bi'otinyl-dl-aspartate precipitated as awhite solid.

Anal. Calcd. for CmHaNaOeS: C, 52.03; H, 7.04; N, "10.11., 'Found: C,52.10; H, 7.12; N, 10.14.

Example 5 "94 mg. of biotin was converted to biotin acid chloride bytreating it with thionyl chloride. To the biotin acid chloride thusobtained was added a solution of 109 mg. of ethyl l(+) glutamatehydrochloride in 2 ml. of pyridine. After 1 /2 hours the pyridine wasevaporated in vacuo and ethyl biotihyl l(+)glutamate remained as an oil.The oil was dissolved in chloroform, Washed successively with diluteacid, bicarbonate, and water. Thechloroform solution was dried andevaporated in ,vacuo leaving ethyl b iotinyl l(+)glutamate as a ol'earoil. White crystalline ethyl bi- 4 otinyl l(+) glutamate wasprecipitated by dissolving the oil in chloroform and diluting withether.

Anal. Calcd. for C19H31N3O6S: C, 53.13; H, 7.28. Found: C, 52.97; H,7.22.

Modifications may be made in carrying out the present invention withoutdeparting from the spirit and scope thereof and the invention is to belimited only by the appended claims.

We claim: p q

1. A compound of the structure wherein R is a substituted aliphaticamine radical linked to the CO group through the amino nitrogen andwherein the only substituents in said amine radical are selected fromthe class consisting of carboxyiic acid and carboxylic acid estergroups.

2. Biotinylglycine having the structural formula 3. Biotinyl-l(-)leucineethyl ester having the structural formula 0 HN-( 7-NH H H Hz(3-S&H(OHl)4C ONH-CHG on 02115 i 7 -H2-CH(CHa)2 4. Biotinyl -B-alaninehaving the structural formula 1 5. Ethyl biotinyl l(+) glutamate havingthe structural formula:

6. Ethyl biotinyldl-aspartate having the structural formula:

7. The process that comprises condensing a compound selected from thegroup consisting of aliphatic amino acids and aliphatic amino acidesters with a compound of the formula 0 HN-(i-NH Ho -53 Hic-"s- 3 H'oraio0iv wherein R is selected from the class consisting of halogen andal'koxy substituents t0 'fblhl the C01- responding biotinyl aliphaticamino compound having the structural formula H2CS-CH-(CH2)4COR wherein Ris a substituted aliphatic amine radical linked to the -C() groupthrough the amino nitrogen and wherein the only substituents in saidamine radical are selected from the class consisting of carboxylic acidand carboxylic acid ester groups.

8. The process that comprises reacting an aliphatic amino acid with acompound of the structure o HN-CLNH H -OH Hlestncri2 4-ooit' wherein Ris selected from the class consisting of halogen and alkoxy substituentsto form the corresponding biotinyl aliphatic amino acid having theformula 1? HITI-NH HC H Hi7-S-J3H-(CH2)4-COR wherein R. is a substitutedamine radical linked to the --CO-- group through the amino nitrogen andwherein the only substituent in said amine radical are carboxylic acidgroups.

9. The process that comprises reacting an aliphatic amino acid esterwith a compound of the structure HzJJS--JH(CHz)4-COR wherein R isselected from the class consisting of halogen and alkoxy substituents toform the corresponding biotinyl aliphatic amino acid ester having theformula H2ts-oH oH)4o0R wherein R is a substituted aliphatic amineradical linked to the -CO- group through the amino nitrogen and whereinthe only substituent in said amine radical are carboxylic acid estergroups and hydrolyzing the latter compound to l( ltmt. 55 +)gua ae formbiotinyl-l()leucine alkyl ester.

11. The process that comprises reacting biotin acid halide with glycinein the presence of a condensing agent and recovering biotinylglycine.

12. The process that comprises reacting biotin 0 acid halide withp-alanine alkyl ester, recovering biotinyl-B-alanine alkyl ester, andhydrolyzing the latter compound to form biotinyl-[i-alanine.

13. The process that comprises reacting biotinyl acid halide with analkyl l(+) glutamate hydrohalide in the presence of a condensing agentto form alkyl biotinyl 1(+)glutamate.

14. The process that comprises reacting biotinyl acid halide withdl-aspartic dialkyl ester in the presence of a condensing agent to formalkyl 0 biotinyl-dl-aspartate.

15. The process that comprises reacting biotin acid chloride withl()leucine ethyl ester hydrochloride in pyridine at room temperature fora time sufficient to effect condensation, evaporating the reactionmixture, extracting the residual oil thus obtained with chloroform,washing the chloroform with dilute acid, drying the chloroform,evaporating the reaction mixture, extracting with ethyl alcohol andrecovering biotinyll()leucine ethyl ester from the extract.

16. The process that comprises reacting biotin acid chloride withglycine in sodium hydroxide, and acidifying the solution withhydrochloric acid to precipitate biotinylglycine.

17. The process that comprises reacting biotinyl acid chloride withdl-aspartic diethyl ester in pyridine, evaporating the pyridine,extracting the residual oil thus obtained with chloroform, washing thechloroform extract successively with dilute acid, bicarbonate and water,drying the chloroform extract, evaporating the chloroform, extractingthe residue with ethyl alcohol, and adding water to precipitate ethylbiotinyl-d1- aspartate.

18. The process that comprises reacting biotinyl acid chloride withethyl 1(+)glutamate hydrochloride in pyridine, evaporating the pyridine,extracting the residue thus obtained with chloroform, washing thechloroform extract successively with dilute acid, bicarbonate, water,drying the chloroform extract, evaporating chloroform, extracting theresidual oil with chloroform and diluting with ether to precipitateethyl biotinyl DONALD E. WOLF. KARL FOLKERS,

No references cited,

1. A COMPOUND OF THE STRUCTURE